By Valerie Ross
Researchers at Case Western Reserve School of Medicine have identified a new protein biomarker for Creutzfeldt-Jakob disease, a fatal prion disorder that causes rapid mental deterioration.
Transferrin, a protein that transports iron in brain tissue, is present at lower levels in the cerebrospinal fluid of patients with Creutzfeldt-Jakob disease than those suffering from other kinds of dementia, they found.
Currently, postmortem analysis of brain tissue is the only way to definitively diagnose Creutzfeldt-Jakob disease. Two other CSF biomarkers — 14-3-3 and T-tau proteins — are now tested in individuals thought to have the disease, but these proteins are not disease-specific markers since their levels can be elevated in other types of dementia, as well.
While no treatment currently exists for Creutzfeldt-Jakob, diagnosis can ensure that the disease is not spread through contact with infected tissue, such as during medical procedures. Additionally, the ability to diagnose the disease early in its course is vital to researchers working to develop treatments.
"Unless you can diagnose [Creutzfeldt-Jakob disease] early enough, you cannot test any therapeutic strategies, even if you can develop any," said Case Western pathologist Neena Singh, an author of the study, which was published in the March edition of PLoS One.
Singh and her colleagues focused on sporadic Creutzfeldt-Jakob disease, or sCJD. The most common form of the disease, sCJD develops spontaneously, with an average age of onset of 65. To investigate possible biomarkers, the researchers tested CSF samples taken pre-mortem from 99 patients with sCJD, as confirmed by autopsy, and 75 patients with dementias caused by other disorders including Alzheimer's disease and encephalitis.
The research team focused their efforts on transferrin because earlier studies have shown that sCJD alters iron metabolism in the brain. In each sample, Singh and her team evaluated levels of total transferrin and levels of two transferrin isomers – Tf-1 and Tf-β2 – by performing Western blot tests, using Genetex Inc.'s anti-transferrin antibody. They also measured total transferrin levels using Alpha Diagnostic International's Human Transferrin ELISA Kit.
Since the altered iron metabolism seen in sCJD could be due not only to changes in the level of transferrin but to changes in the amount of iron it transports, the researchers also measured the iron saturation level of transferrin in the samples to make sure the saturation levels were equivalent. To compare the new biomarker to the current standard biomarker, T-tau, the researchers measured levels of T-tau using Invitrogen's Human Tau (total) ELISA Kit.
Levels of total transferrin were lower in the CSF of sCJD patients than patients with other types of dementia, the researchers found, as were levels of both Tf-1 and Tf-β2. Of the three proteins, total transferrin provided the highest accuracy as a biomarker, distinguishing sCJD from other dementias with an accuracy of 80 percent, compared to the 70 percent accuracy that Singh and her colleagues obtained with the current gold-standard T-tau biomarker test. The total transferrin test had a sensitivity of 86 percent and a specificity of 72 percent.
The researchers additionally found that when they looked at levels of both transferrin and T-tau together they could distinguish sCJD from other dementias with an accuracy of 86 percent – 85 percent sensitivity and 88 percent specificity. While T-tau is not specific to sCJD, it indicates cell death in the brain, Singh said. “Combining with tau increased the specificity significantly because now we have a marker for cell death combined with a marker specific to the disease.”
Anders Skinningsrud, a clinical researcher at Akershus University Hospital in Norway who has investigated CSF biomarkers for sCJD, suggested that, while further research is needed to verify these results, Singh's study was well-performed and is a positive step toward diagnosis.
"As long as the pathological prion protein [underlying sCJD] cannot be detected routinely in CSF, the addition of CSF-transferrin as a marker for prion diseases may increase the pre-mortal diagnostic accuracy of sCJD," he wrote in an e-mail to ProteoMonitor.
To diagnose sCJD, a test must measure CSF transferrin levels rather than blood transferrin levels because iron metabolism — and therefore transferrin — is only affected in the brain, not throughout the body, Singh said. However, since blood transferrin levels are routinely checked during other tests, she said, "we can very easily modify that instrument to check for transferrin in CSF, and it can be a very rapid test."
Transferrin is also relatively abundant in CSF compared to T-tau and 14-3-3 proteins, present in concentrations of micrograms per mL versus picograms per mL for the latter two. This makes it possible to test for transferrin even in small samples, Singh noted, adding to its potential utility as a biomarker.
Skinningsrud agreed that the ease of measuring transferrin would make it an improvement over current diagnostic tests. "The possibility exists that CSF-transferrin might replace 14-3-3-protein, which will be a good thing because CSF-transferrin is more easy to do routinely," he said.
In follow-up studies, Singh hopes to demarcate a cutoff value for CSF transferrin that clinicians can use to diagnose sCJD.
"We want to collect normal samples from non-dementia cases, cases like lymphoma or tumor or some other disease of the brain, and establish the normal value of transferrin in those CSF samples," she said. "Then, centers around the world can run the test and analyze it relative to that value, and if the levels were lower they could know if it was CJD."
She also hopes to combine a test for CSF transferrin with other tests of iron metabolism to see if that will further improve the sensitivity and specificity of diagnosis. Singh said she does not currently have plans to commercialize the test.
In addition to their main findings, Singh and her team also noticed that CSF from sCJD patients showed less of a decrease in transferrin levels when compared to Alzheimer's disease patients than patients with other types of dementia — 39 percent compared to 49 percent — suggesting that Alzheimer's patients may also have slightly decreased levels of CSF transferrin.
While previous studies have indicated that brain iron metabolism may be affected by Alzheimer's, "we were surprised to see that Alzheimer's disease cases show it," Singh said.
She added that the researchers hope to continue their work "and see if we can establish something for Alzheimer's disease, also."
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